PROJECT SUMMARY The continued and inevitable emergence of antibiotic resistance demands a vigorous and sustained effort to identify fundamentally new targets and strategies for innovative antimicrobial therapeutics. Antibiotic-resistant enterococci are major causes of hospital-acquired infections. Enterococci are successful hospital-acquired pathogens in part because of their intrinsic resistance to commonly used antibiotics that target the bacterial cell envelope, such as cephalosporins. However, many questions remain regarding the genetic and biochemical basis for cephalosporin resistance in enterococci. In preliminary studies we identified two widely conserved genes encoding ?conserved hypothetical? proteins of unknown function (termed IreB and IreC) that modulate intrinsic enterococcal cephalosporin resistance. IreB acts as a negative regulator of cephalosporin resistance, while IreC promotes resistance. However, the mechanisms by which IreB and IreC influence cephalosporin resistance are unknown, and few clues are available to guide the development of hypotheses to explain their roles. The research proposed here is designed to elucidate new insights into the roles of IreB and IreC in the biological processes that drive enterococcal cephalosporin resistance. To do so, we will use unbiased, genome-wide genetic approaches to identify cellular factors that influence cephalosporin resistance in cooperation with IreB and IreC. By doing so, the research proposed here will provide new insights into the fundamental biological processes that drive key antibiotic resistance in enterococci and may define new targets for innovative therapeutics designed to impair enterococcal antibiotic resistance.